Protein engineering of sulfhydryl groups and methionine in mungbean [vigna radiata (L.) R. Wilczer] vicilin and effects on its nutritional and functional properties
2007
Torio, M.A.O.
The 8S-alpha globulin, the major storage protein of mungbean, was engineered to introduced sulfhydryl groups and disulfide bond to improve its structural stability and functional properties and methionine residues to improve nutritional quality. Using site-directed mutagenesis, mutants F59C, 199C and A213C were designed and prepared with free sulfhydryl group, 199C/A213C with disulfide bond and F59C/199C/A213C with both a free sulfhdryl group and disulfide 8S-a globulin. Mutants 199C/A213C and F59C/199C/A213C formed a disulfide bond as expected which was confirmed by the Ellman method. Mutants with introduced disulfide bond exhibited greater resistance to enzymatic digestion compared to the wild type (WT). All mutants showed greater hardness of heat-induced gels than WT, specially 199C/A213C and F59C/199C/A213C mutants at 2% protein concentration. The results indicate that increasing sulfhydryl groups and disulfide bonds increases structural stability. Eight methionine-mutants, IIa, IVa, IIa+b, IVa+b,II+4, IV+4, IVa+b=IIa and IVa+b+IIa+b, were designed and prepared using site-directed mutagenesis. Mutants IVa, IIa, II+4, IV+4, IIa+b, IVa+b+IIa and IVa+b+IIa+b contain 2,3,4,5,8 and 10 methionine residues, respectively. Mutants with introduced methionine exhibited greater stability in terms of thermal denaturation but similar pattern to enzymatic digestion compared to the Wild Type (WT). All mutants showed greater emulsifying ability than Wt, specially IVa+b+IIa+b. Based on allergenicity prediction programs (BLASP and ADFS), WT and all mutants had no allergenic potential. The result indicate that increasing methionine content increases structural stability in terms of thermal denaturation and emulsifying ability.
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